Development of a multicomplex nanostructured system associated with the use of RNAi to control tomato chlorosis virus (ToCV)

• Grant number: 23/07905-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: April 01, 2024
• End date: March 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Leonardo Fernandes Fraceto
• Grantee: Felipe Franco de Oliveira
• Host Institution: Instituto de Ciência e Tecnologia. Universidade Estadual Paulista (UNESP). Campus de Sorocaba. Sorocaba , SP, Brazil
• Associated research grant: 21/10639-5 - Center for Research on Biodiversity Dynamics and Climate Change, AP.CEPID
• Associated scholarship(s): 25/00937-0 - Comparative tracking and effectiveness of positively and negatively charged dsRNA zein nanoparticles to suppress the potato virus Y (PVY), BE.EP.PD

Abstract

Among the problems that affect the tomato crop (Solanum lycopersicum), the incidence of viral diseases is a major concern for growers. Those caused by viruses belonging to the genus crinivirus, such as tomato chlorosis virus (ToCV), are globally important, mainly to high incidences and severity, causing losses in production. ToCV is transmitted by several species of Bemisia tabaci, with a wide range of cultivated and weed hosts. Currently, the main management method is the use of chemical insecticides to control the vector. However, despite the effectiveness in controlling the insect, insecticides are not able to prevent the virus transmission, resulting in epidemics and increasing amounts of chemicals. The global demand for healthy food and in the context of climate change, where it is believed that plant diseases can become increasingly significant, new solutions for management become essential. Thus, the use of interfering RNA mechanisms (RNAi), a natural mechanism of plants against pathogens, has been recently gaining ground as a new tool for the management of plant viruses based on the strategy of gene silencing. The application of dsRNA is a common way to start the RNAi process in plants, it is based on a region of interest of the virus genome. However, despite the effectiveness of control, these oligonucleotides are easily degraded, have little persistence in the plant, and are distributed throughout the plant irregularly. In this context, nanotechnology emerges as a potential tool to improve the performance of active ingredients, especially in terms of efficiency in application and use, as well as in reducing the volume of active ingredients used in the field. The objective of this project is to develop a biodegradable multicomplex nanostructured system associated with the use of dsRNA as a trigger of the RNAi process to control tomato yellowing caused by ToCV. First, potential base regions for dsRNA synthesis will be selected through the generation of profiles of small RNAs from ToCV infections of tomato plants. Then, the three most frequent regions will be selected, which will serve as a template for dsRNA synthesis. The three dsRNAs will be tested for efficacy and duration of control. The dsRNAs will also be subjected to distribution and persistence tests in plants via RT-qPCR. Subsequently, nanoformulations in two different matrices (Zein and LDH) will be synthesized with the best performance region of dsRNA and mixed (NANOdsRNA). The morphological and physical-chemical characteristics will then be measured to detect possible effects of the mixture. Once the formulation is established, efficacy tests against ToCV in tomato plants will be carried out again, evaluating the infection rate and duration of protection. To understand changes in distribution dynamics, tests will be performed with NANOdsRNA applied to plants, using RT-qPCR. An additional test will be carried out by adding the Cy-3 marker, which will be observed for distribution, through confocal microscopy. With this project, we hope to develop a new tool to control ToCV in tomato plants, through the combination of two modern and sustainable technologies (RNAi and nanotechnology), pioneering the combination of two nanoformulation matrices for better delivery of the active, in addition to providing new knowledge about their behavior in planta.